Etherlipid containing multiple lipid liposomes

ABSTRACT

Liposomes are provided which contain etherlipids having the formula: ##STR1## as well as an unsaturated or partially unsaturated phospholipid, a sterol, and a phosphatidylethanolamine-based headgroup-derivatized lipid. These liposomes are useful in a variety of therapeutic regimens, including the treatment of cancers and inflammatory disorders.

This application is a CIP of Ser. No. 08/602,669 filed Feb. 16, 1996,now U.S. Pat. No. 5,762,958.

Etherlipids are synthetic analogues of platelet activating factor (PAF;1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), an effector generallybelieved to be involved in a variety of physiological processes, such asinflammation, the immune response, allergic reactions and reproduction.Etherlipids have been shown to be effective antitumor agents in animals,and are believed to be selectively cytotoxic to a broad variety ofcancer cells (see, for example, Dietzfelbinger et al. (1993); Zeisig etal. (1993); Powis et al. (1990); Berdel (1991); Bhatia and Hadju (1991);Reed et al. (1991); Workman (1991); Workman et al. (1991); Bazill andDexter (1990); Berdel (1990); Counsell et al. (1990); Tritton andHickman (1990); Muschiol et al. (1990); Layton et al. (1980); Runge etal. (1980); Great Britain Patent No. 1,583,661; U.S. Pat. No.3,752,886). Etherlipids have also been shown to be antimetastatic andanti-invasive, and to be capable of cell differentiation induction.

Mechanisms of etherlipid cytotoxicity, while not definitivelyestablished, appear to involve action at, and possible disruption of,the cell membrane. The selective cytotoxicity of etherlipids may involveintracellular accumulation and differential activity of alkyl cleavageenzymes. Etherlipids may also be selective inhibitors ofphosphatidylinositol phospholipase C and protein kinase C activities, aswell as of phosphatidylcholine biosynthesis. Hence, etherlipids arepotentially quite useful as therapeutic agents. However, theiradministration can also lead to hemolysis, hepatic dysfunction andgastrointestinal disorders. Applicants have found that certain liposomalformulations of etherlipids can buffer these toxicities withoutinhibiting anticancer efficacy, and thereby can provide a moretherapeutically useful basis for etherlipid administration.

SUMMARY OF THE INVENTION

This invention provides a liposome comprising a bilayer having a lipidcomponent which comprises: (a) an unsaturated or partially unsaturatedphospholipid; (b) a sterol; (c) a headgroup derivatized lipid containinga phosphatidylethanolamine linked to a moiety selected from the groupconsisting of dicarboxylic acids, polyethylene glycols, gangliosides andpolyalkylethers; and, (d) an etherlipid. The headgroup-derivatized lipidcomprises from about 5 mole percent to about 20 mole percent of thebilayer's lipid component; the etherlipid comprises from about 10 molepercent to about 30 mole percent of the lipid component.

Preferably, the phospholipid is dioleoyl phosphatidylcholine ("DOPC"),the sterol is cholesterol ("chol"), the headgroup-derivatized lipid isdioleoyl phosphatidylethanolamine-glutaric acid ("DOPE-GA") and theetherlipid is: ##STR2## also known as "EL-18, " "ET-18-OCH₃," or"edelfosine"). Most preferably, presently, the liposome is a unilamellarliposome having a diameter of from greater than about 50 nm to less thanabout 200 nm and comprises a bilayer having a lipid component whichcomprises about 20 mole percent of the etherlipid, about 10 mole percentof the headgroup-derivatized lipid, about 30 mole percent cholesteroland about 40 mole percent dioleoyl phosphatidylethanolamine. Theliposome can further comprise an additional bioactive agent, that is anagent in addition to the etherlipid.

Also provided herein is a pharmaceutical composition comprising apharmaceutically acceptable carrier and the liposomes of this invention.Further provided is a method of treating a mammal afflicted with acancer, including, but not limited to: a lung, brain, colon, ovarian orbreast cancers, the method comprising administering the compositions ofthis invention to the mammal, in an amount containing an anticancereffective amount of the etherlipid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Time Course of Carboxyfluorescein Leakage from LiposomalEdelfosine Formulations Incubated at 48 deg. Celsius in PBS. ELL 28(uppermost curve, "ELL" indicating "etherlipid liposome"): Distearoylphosphatidylcholine ("DSPC"); cholesterol ("CHOL"); dioleoylphosphatidylethanolamine-glutaric acid ("DOPE-GA"); edelfosine "EL,"standing for "etherlipid" (the respective molar ratio of these lipidcomponents being 4:3:1:2); ELL 30 (second from top curve):EPC:CHOL:DOPE-GA:EL (4:3:1:2); ELL 25 (middle curve):DOPE:CHOL:DOPE-GA:EL (3:3:1:3); ELL 12 (second from bottom curve):DOPC:CHOL:DOPE-GA:EL (4:3:1:2); and, ELL 20 (bottom curve):DOPE:CHOL:DOPE-GA:EL (4:3:1:2). Y-axis: % CF Leakage; x-axis: time(seconds).

FIG. 2. Comparison of Hemolytic Activity and CF Leakage in EtherlipidLiposomes. From top-to-bottom: ELL 20-ELL 12-ELL 25-ELL 30-ELL 28(y=34231x⁻².1614 ; R² =0.96). Y-axis: HI₁₀ ; x-axis: % CF leakage uponincubation in PBS.

FIG. 3. Stability of Etherlipid Liposomal Formulations on Incubation in0.5% Serum at 37 Degrees Celsius. Y-axis: time (minutes); x-axis (fromleft-to-right): ELL 28, ELL 40, ELL 30; ELL 25; ELL 12; ELL 20. Inset:Y-axis: time (minutes); x-axis: ELL 28, ELL 40, ELL 30.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a liposome comprising a bilayer having a lipidcomponent which comprises: (a) an unsaturated or partially unsaturatedphospholipid; (b) a sterol; (c) a headgroup derivatized lipid containinga phosphatidylethanolamine and a moiety selected from the groupconsisting of dicarboxylic acids, gangliosides, polyethylene glycols andpolyalkylethers, which headgroup-derivatized lipid comprises from about5 mole percent to about 20 mole percent of the bilayer's lipidcomponent; and, (d) an etherlipid having the following formula: ##STR3##the etherlipid comprising from greater than about 10 mole percent, toless than about 30 mole percent, of the bilayer's lipid component.

"Liposomes" are self-assembling structures comprising one or more lipidbilayers, each of which surrounds an aqueous compartment and comprisestwo opposing monolayers of amphipathic lipid molecules. Amphipathiclipids comprise a polar (hydrophilic) headgroup region covalently linkedto one or two non-polar (hydrophobic) acyl chains. Energeticallyunfavorable contacts between the hydrophobic acyl chains and the aqueousmedium are generally believed to induce lipid molecules to rearrangesuch that the polar headgroups are oriented towards the aqueous mediumwhile the acyl chains reorient towards the interior of the bilayer. Anenergetically stable structure is formed in which the acyl chains areeffectively shielded from coming into contact with the aqueous medium.

Liposomes can have a single lipid bilayer (unilamellar liposomes,"ULVs"), or multiple lipid bilayers (multilamellar liposomes, "MLVs"),and can be made by a variety of methods (for a review, see, for example,Deamer and Uster (1983)). These methods include without limitation:Bangham's methods for making multilamellar liposomes (MLVs); Lenk's,Fountain's and Cullis' methods for making MLVs with substantially equalinterlamellar solute distribution (see, for example, U.S. Pat. Nos.4,522,803, 4,588,578, 5,030,453, 5,169,637 and 4,975,282); andPapahadjopoulos et al.'s reverse-phase evaporation method (U.S. Pat. No.4,235,871) for preparing oligolamellar liposomes. ULVs can be producedfrom MLVs by such methods as sonication (see Papahadjopoulos et al.(1968)) or extrusion (U.S. Pat. No. 5,008,050 and U.S. Pat. No.5,059,421). The etherlipid liposome of this invention can be produced bythe methods of any of these disclosures, the contents of which areincorporated herein by reference.

Various methodologies, such as sonication, homogenization, French Pressapplication and milling can be used to prepare liposomes of a smallersize from larger liposomes. Extrusion (see U.S. Pat. No. 5,008,050) canbe used to size reduce liposomes, that is to produce liposomes having apredetermined mean size by forcing the liposomes, under pressure,through filter pores of a defined, selected size. Tangential flowfiltration (see WO89/008846), can also be used to regularize the size ofliposomes, that is, to produce liposomes having a population ofliposomes having less size heterogeneity, and a more homogeneous,defined size distribution. The contents of these documents areincorporated herein by reference. Liposome sizes can also be determinedby a number of techniques, such as quasi-electric light scattering, andwith equipment, e.g., Nicomp® particle sizers, well within thepossession of ordinarily skilled artisans.

The liposomes of this invention can be unilamellar or multilamellar.Preferably the liposomes are unilamellar and have diameters of less thanabout 200 nm, more preferably, from greater than about 50 nm to lessthan about 200 nm; such liposomes are preferably produced by a methodcomprising the steps of: dissolving lipids in a suitable organic solventso as to establish a lipidic solution; removing the organic solvent fromthe resulting lipidic solution; adding an aqueous solution so as to formliposomes; and, then extruding the resulting liposomes through asuitable filter.

Liposomes of the 50-200 nm size are preferred because they generallybelieved to circulate longer in mammals than do larger liposomes, whichare more quickly recognized by the mammals' reticuloendothelial systems("RES"), and hence, more quickly cleared from the circulation. Longercirculation can enhance therapeutic efficacy by allowing more liposomesto reach their intended site of actions, e.g., tumors or inflammations.Small unilamellar liposomes, i.e., those generally less than 50 nm indiameter, carry amounts of bioactive agents which may be, in some cases,too low to be of sufficient therapeutic benefit.

R₁ of the etherlipid, the chain attached at the carbon #1 position ofits glycerol backbone by way of an oxygen, has the formula Y₁ Y₂. Y₂ isCH₃ or CO₂ H, but preferably is CH₃. Y₁ is --(CH₂)_(n1) (CH═CH)_(n2)(CH₂)_(n3) (CH═CH)_(n4) (CH₂)_(n5) (CH═CH)_(n6) (CH₂)_(n7) (CH═CH)_(n8)(CH₂)_(n9) ; the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer offrom 3 to 23; that is, the acyl chain is from 4-24 carbon atoms inlength. n1 is equal to zero or is an integer of from 1 to 23; n3 isequal to zero or is an integer of from 1 to 20; n5 is equal to zero oris an integer of from 1 to 17; n7 is equal to zero or is an integer offrom 1 to 14; n9 is equal to zero or is an integer of from 1 to 11; andeach of n2, n4, n6 and 8 is independently equal to zero or 1.

The hydrocarbon chain is preferably saturated, that is, it preferablyhas no double bonds between adjacent carbon atoms, each of n2, n4, n6and n8 then being equal to zero. Accordingly, Y₁ is preferably(CH₂)_(n1). More preferably, R₁ is (CH₂)_(n1) CH₃, and most preferably,is (CH₂)₁₇ CH₃. Alternatively, the chain can have one or more doublebonds, that is, it can be unsaturated, and one or more of n2, n4, n6 andn8 can be equal to 1. For example, when the unsaturated hydrocarbon hasone double bond, n2 is equal to 1, n4, n6 and n8 are each equal to zeroand Y₁ is (CH₂)_(n1) CH═CH(CH₂)_(n3). n1 is equal to zero or is aninteger of from 1 to 21, and n3 is also zero or is an integer of from 1to 20, at least one of n1 or n3 not being equal to zero.

Z is oxygen, sulfur, NH, or --NHC(O)--, Z then being connected to themethyl group by way of either the nitrogen or carbonyl carbon. Z canalso be --OC(O)--, it then being connected to the methyl group by way ofeither the oxygen or carbonyl carbon atom. Preferably, Z is O;accordingly, this invention's glycerol-based etherlipids preferably havea methoxy group at the sn-2 position of their glycerol backbone.

R₂ is an alkyl group, or a halogen-substituted alkyl group, having theformula (C(X₁)_(n10) (X₂)_(n11))_(n12) CX₃ X₄ X₅, wherein each of X₁,X₂, X₃, X₄, and X₅ is independently hydrogen or a halogen, but ispreferably hydrogen. n10 is equal to zero, 1 or 2; n11 is equal to zero,1, or 2; and n12 is equal to zero or an integer of from 1 to 23, but ismost preferably, zero, R₂ then being CX₃ X₄ X₅. X₃, X₄, and X₅ are mostpreferably H, R₂ then being CH₃. Accordingly, the etherlipid preferablyhas a methyl group attached to its carbon #2. However, R₂ can then alsobe CH₂ F, CHF₂ or CF₃. When n12 is not zero, the sum of n10+n11 is equalto 2, n12 is preferably equal to 1, and R₂ is preferably CH₂ CH₃, CH₂CF₃ or CF₂ CF₃.

Most preferably, the etherlipid is one in which Y₂ is CH₃, R₁ is(CH₂)_(n1) CH₃, R₂ is CH₃ and Z is O. The preferred etherlipid istherefore: ##STR4## that is,1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine ("ET-18-OCH₃ " or"edelfosine").

The unsaturated or partially unsaturated phospholipid, having two acylchains, at least one of which has at least one double bond betweenadjacent carbon atoms, is preferably a phosphatidylcholine ("PC"). Mostpreferably, presently, the PC is dioleoyl phosphatidylcholine ("DOPC").The liposome's lipid bilayer also contains a sterol, which generallyaffects the fluidity of lipid bilayers (see, for example, Lewis andMcElhaney (1992) and Darnell et al. (1986)) Accordingly, sterolinteractions with surrounding hydrocarbon chains generally inhibitemigration of these chains from the bilayer. The sterol of the liposomesof this invention is preferably, but not necessarily, cholesterol, andcan also be a variety of other sterolic compounds.

A "headgroup-derivatized" lipid is a lipid which, when present in aliposomal lipid bilayer with an etherlipid, can buffer the toxicity ofthe etherlipid. That is, the derivatized lipid can decrease theetherlipid's toxicity, such that it is generally less toxic than thefree form of the etherlipid. Headgroup-derivatized lipids generally areamphipathic lipids comprising hydrophobic acyl chains, and aphosphorylethanolamine group to which a suitable chemical moiety hasbeen attached. Acyl chains are those which can adopt compatible packingconfigurations with the hydrophobic portions of other lipids present inthe bilayer, and which can interact with an etherlipid such that releaseof the etherlipid from the bilayer is inhibited and etherlipid toxicityis buffered; these are saturated or unsaturated, straight-chained orbranched, and typically contain from 4 to 24 carbon atoms in a straightchain. Preferred acyl chains are palmitate or oleate chains; hencepreferred headgroup-modified lipids are dipalmitoylphosphatidylethanolamine ("DPPE"), palmitoyloleoylphosphatidylethanolamine ("POPE") or dioleoyl phosphatidylethanolamine("DOPE"); most preferably, presently, the lipid is DOPE.

Preferably, the headgroup derivatized lipid used herein is dipalmitoylphosphatidylethanolamine ("DPPE"), palmitoyloleoylphosphatidylethanolamine ("POPE") or dioleoyl phosphatidylethanolamine("DOPE"). Most preferably, presently, the lipid is DOPE. Chemicalmoieties suitable for attachment to such lipids are those, such asdicarboxylic acids, gangliosides, polyethylene glycols, polyalkyl ethersand the like, which can be attached to the amino group of aphosphorylethanolamine, and which give rise to lipids having toxicitybuffering, circulation-enhancing properties. Means of identifyingsuitable chemical moieties, for example by subjecting derivatized lipidsto in vitro and in vivo toxicity testing, are well known to, and readilypracticed by, ordinarily skilled artisans given the teachings of thisinvention. Means of attaching chemical moieties tophosphorylethanolamine groups are also well known to, and readilypracticed by, ordinarily skilled artisans.

Toxicity buffering capacities of headgroup-derivatized lipids can bedetermined by a number of in vitro and in vivo testing methods wellknown to, and readily practiced by, ordinarily skilled artisans, giventhe teachings of this invention. For example, etherlipid-induced redblood cell (RBC) hemolysis can be examined in vitro by combining anetherlipid with an RBC suspension, incubating the combination, and thenquantitating the percentage of RBC lysis.

Toxicity-buffering can also be assessed by determining the etherlipid'stherapeutic window "TW," which is a numerical value derived from therelationship between the compound's induction of hemolysis and itsability to inhibit the growth of tumor cells. TW values are determinedin accordance with the formula HI₁₅ /GI₅₀ (wherein "HI₅ " equals theconcentration of compound inducing the hemolysis of 5% of the red bloodcells in a culture, and wherein "GI₅₀ " equals the dose of compoundinducing fifty percent growth inhibition in a population of cellsexposed to the agent). The higher an agent's HI₅ value, the lesshemolytic is the agent--higher HI₅ 's mean that greater concentrationsof compound are required to be present in order for the compound toinduce 5% hemolysis. Hence, the higher its HI₅, the more therapeuticallybeneficial is a compound, because more of it can be given beforeinducing the same amount of hemolysis as an agent with a lower HI₅. Bycontrast, lower GI₅₀ 's indicate better therapeutic agents--a lower GI₅₀value indicates that a lesser concentration of an agent is required for50% growth inhibition. Accordingly, the higher is its HI₅ value and thelower is its GI₅₀ value, the better are a compound's agent's therapeuticproperties.

Generally, when a bioactive agent's TW is less than 1, it cannot be usedeffectively as a therapeutic agent. That is, the agent's HI₅ value issufficiently low, and its GI₅₀ value sufficiently high, that it isgenerally not possible to administer enough of the agent to achieve asufficient level of tumor growth inhibition without also attaining anunacceptable level of hemolysis. Etherlipid liposomes having bilayersthat also comprise headgroup-derivatized lipids have TW's of greaterthan 1. Preferably, the TW of an etherlipid in a liposomal bilayer alsocomprising a headgroup-derivatized lipid is greater than about 1.5, morepreferably, greater than about 2, and still more preferably, greaterthan about 3.

Headgroup-derivatized lipids can also be circulation-enhancing lipids,that is, the modifications directed to lipid toxicity buffering can alsoafford circulation enhancement. Accordingly, headgroup-derivatizedlipids can inhibit clearance of liposomes from the circulatory systemsof animals to which they have been administered. Liposomes are generallybelieved to be cleared from an animal's body by way of itsreticuloendothelial system (RES). Avoiding RES clearance means that thefrequency of liposome administration can be reduced, and that less of aliposome-associated bioactive agent need be administered to achievedesired serum levels of the agent. Enhanced circulation times can alsoallow targeting of liposomes to non-RES containing tissues.

Liposome outer surfaces are believed to become coated with serumproteins, such as opsonins, in animals' circulatory systems. Withoutintending in any way to be limited by theory, it is believed thatliposome clearance can be inhibited by modifying the outer surface ofliposomes such that binding of serum proteins thereto is generallyinhibited. Effective surface modification, that is, alterations to theouter surfaces of liposomes which result in inhibition of opsonizationand RES uptake, is believed to be accomplished by incorporating intoliposomal bilayers lipids whose polar headgroups have been derivatizedby attachment thereto of a chemical moiety which can inhibit the bindingof serum proteins to liposomes such that the pharmacokinetic behavior ofthe liposomes in the circulatory systems of animals is altered (see,e.g., Blume et al. (1993); Gabizon et al. (1993); Park et al. (1992);Woodle et al. U.S. Pat. No. 5,013,556; and, U.S. Pat. No. 4,837,028).

Presently, dicarboxylic acids, such as glutaric, sebacic, succinic andtartaric acids, are preferred components of headgroup-derivatizedlipids. Most preferably, the dicarboxylic acid is glutaric acid ("GA").Accordingly, suitable headgroup-derivatized lipids includephosphatidylethanolamine-dicarboxylic acids such as dipalmitoylphosphatidylethanolamine-glutaric acid ("DPPE-GA"), palmitoyloleoylphosphatidylethanolamine-glutaric acid ("POPE-GA") and dioleoylphosphatidylethanolamine-glutaric acid ("DOPE-GA"). Most preferably, thederivatized lipid is DOPE-GA.

The liposomes of this invention can comprise one or more additionallipids, that is, lipids in addition to the phosphatidylcholine, sterol,headgroup-derivatized lipid and etherlipid already present in theliposomes' bilayers. Additional lipids are selected for their ability toadapt compatible packing conformations with the other lipid componentsof the bilayer such that the lipid constituents are tightly packed, andrelease of the lipids from the bilayer is inhibited. Lipid-based factorscontributing to compatible packing conformations are well known toordinarily skilled artisans and include, without limitation, acyl chainlength and degree of unsaturation, as well as the headgroup size andcharge. Accordingly, suitable additional lipids, including variousphosphatidylethanolamines ("PE's") such as egg phosphatidylethanolamine("EPE") or dioleoyl phosphatidylethanolamine ("DOPE") can be selected byordinarily skilled artisans without undue experimentation.

Preferred embodiments of this invention have the unsaturated orpartially unsaturated phospholipid being the phosphatidylcholine DOPC,the sterol being cholesterol ("chol"), the headgroup-derivatized lipidbeing DOPE-GA and the etherlipid being ET-18-OCH₃. Most preferably,presently, the liposome comprises DOPC, chol, DOPE-GA and ET-18-O--CH₃in a respective molar ratio of 4:3:1:2, wherein DOPC comprises 40 mole %of the liposomes' bilayers, chol 30% mole, DOPE-GA 10 mole % and theetherlipid 20 mole %. Preferably, the liposomes are unilamellar and havean average diameter of from about 50 nm to about 200 nm, "average"meaning that the median diameter of a population of this invention'sliposomes is between about 50 and 200 nm.

The liposome can comprise an additional bioactive agent, that is, abioactive agent in addition to the etherlipid. A "bioactive agent" isany compound or composition of matter that can be administered toanimals, preferably humans. Such agents can have biological activity inanimals; the agents can also be used diagnostically in the animals.Bioactive agents which may be associated with liposomes include, but arenot limited to: antiviral agents such as acyclovir, zidovudine and theinterferons; antibacterial agents such as aminoglycosides,cephalosporins and tetracyclines; antifungal agents such as polyeneantibiotics, imidazoles and triazoles; antimetabolic agents such asfolic acid, and purine and pyrimidine analogs; antineoplastic agentssuch as the anthracycline antibiotics and plant alkaloids; sterols suchas cholesterol; carbohydrates, e.g., sugars and starches; amino acids,peptides, proteins such as cell receptor proteins, immunoglobulins,enzymes, hormones, neurotransmitters and glycoproteins; dyes;radiolabels such as radioisotopes and radioisotope-labeled compounds;radiopaque compounds; fluorescent compounds; mydriatic compounds;bronchodilators; local anesthetics; and the like.

Liposomal bioactive agent formulations can enhance the therapeutic indexof the bioactive agent, for example by buffering the agent's toxicity.Liposomes can also reduce the rate at which a bioactive agent is clearedfrom the circulation of animals. Accordingly, liposomal formulation ofbioactive agents can mean that less of the agent need be administered toachieve the desired effect. Additional bioactive agents preferred forthe liposome of this invention include antimicrobial, anti-inflammatoryand antineoplastic agents, or therapeutic lipids, for example,ceramides. Most preferably, the additional bioactive agent is anantineoplastic agent.

Liposomes can be loaded with one or more biologically active agents bysolubilizing the agent in the lipid or aqueous phase used to prepare theliposomes. Alternatively, ionizable bioactive agents can be loaded intoliposomes by first forming the liposomes, establishing anelectrochemical potential, e.g., by way of a pH gradient, across theoutermost liposomal bilayer, and then adding the ionizable agent to theaqueous medium external to the liposome (see Bally et al. U.S. Pat. No.5,077,056 and WO86/01102).

The liposome of this invention can be dehydrated, stored and thenreconstituted such that a substantial portion of its internal contentsare retained. Liposomal dehydration generally requires use of ahydrophilic drying protectant such as a disaccharide sugar at both theinside and outside surfaces of the liposome bilayers (see U.S. Pat. No.4,880,635). This hydrophilic compound is generally believed to preventthe rearrangement of the lipids in the liposome, so that the size andcontents are maintained during the drying procedure and throughsubsequent rehydration. Appropriate qualities for such dryingprotectants are that they be strong hydrogen bond acceptors, and possessstereochemical features that preserve the intramolecular spacing of theliposome bilayer components. Alternatively, the drying protectant can beomitted if the liposome preparation is not frozen prior to dehydration,and sufficient water remains in the preparation subsequent todehydration.

Also provided herein is a pharmaceutical composition comprising apharmaceutically acceptable carrier and the liposome of this invention."Pharmaceutically acceptable carriers" as used herein are those mediagenerally acceptable for use in connection with the administration oflipids and liposomes, including liposomal bioactive agent formulations,to animals, including humans. Pharmaceutically acceptable carriers aregenerally formulated according to a number of factors well within thepurview of the ordinarily skilled artisan to determine and account for,including without limitation: the particular liposomal bioactive agentused, its concentration, stability and intended bioavailability; thedisease, disorder or condition being treated with the liposomalcomposition; the subject, its age, size and general condition; and thecomposition's intended route of administration, e.g., nasal, oral,ophthalmic, topical, transdermal, vaginal, subcutaneous, intramammary,intraperitoneal, intravenous, or intramuscular (see, for example, Nairn(1985)). Typical pharmaceutically acceptable carriers used in parenteralbioactive agent administration include, for example, D5W, an aqueoussolution containing 5% weight by volume of dextrose, and physiologicalsaline. Pharmaceutically acceptable carriers can contain additionalingredients, for example those which enhance the stability of the activeingredients included, such as preservatives and anti-oxidants.

Further provided is a method of treating a mammal afflicted with acancer, e.g., a brain, breast, lung, colon or ovarian cancer, or aleukemia, lymphoma, sarcoma, carcinoma, which comprises administeringthe pharmaceutical composition of this invention to the mammal,etherlipids being believed to be selectively cytotoxic to tumor cells.Generally, liposomal etherlipids can be used to treat cancers treatedwith free, that is, nonliposomal, etherlipids. However, encapsulation ofan etherlipid in a liposome can enhance its therapeutic index, andtherefore make the liposomal etherlipid a more effective treatment.

An amount of the composition comprising an anticancer effective amountof the etherlipid, typically from about 0.1 to about 1000 mg of thelipid per kg of the mammal's body, is administered, preferablyintravenously. For the purposes of this invention, "anticancer effectiveamounts" of liposomal etherlipids are amounts effective to inhibit,ameliorate, lessen or prevent establishment, growth, metastasis orinvasion of one or more cancers in animals to which the etherlipids havebeen administered. Anticancer effective amounts are generally chosen inaccordance with a number of factors, e.g., the age, size and generalcondition of the subject, the cancer being treated and the intendedroute of administration, and determined by a variety of means, forexample, dose ranging trials, well known to, and readily practiced by,ordinarily skilled artisans given the teachings of this invention.Antineoplastic effective amounts of the liposomal etherlipid of thisinvention are about the same as such amounts of free, nonliposomal,etherlipids, e.g., from about 0.1 mg of the etherlipid per kg of bodyweight of the mammal being treated to about 1000 mg per kg.

Preferably, the liposome administered is a unilamellar liposome havingan average diameter of from about 50 nm to about 200 nm. The anti-cancertreatment method can include administration of one or more bioactiveagents in addition to the liposomal etherlipid, these additional agentspreferably, but not necessarily, being included in the same liposome asthe etherlipid. The additional bioactive agents, which can be entrappedin liposomes' internal compartments or sequestered in their lipidbilayers, are preferably, but not necessarily, anticancer agents orcellular growth promoting factors.

This invention will be better understood from the following examples.However, those of ordinary skill in the art will readily understand thatthese examples are merely illustrative of the invention as defined inthe claims which follow thereafter.

EXAMPLES Example 1

Preparation

Liposomes were prepared with edelfosine (ET-18-O--CH₃, 5 mg/ml), variousother lipids obtained from Avanti Polar Lipids, Birmingham, Ala., andcholesterol (Sigma Chemical Co.). Briefly, the lipids were dissolved inan organic solvent, such as chloroform, at various mole ratios. Theorganic solvent was then removed, and the dried lipids were rehydrated,e.g., with Dulbecco's phosphate-buffered saline (D-PBS) (Gibco BRL LifeTechnologies, Grand Island, N.Y.). The resulting liposomes were extrudedthrough 0.1 micron Nuclepore® filters (see, for example, Mayer et al.,1985). Liposome sizes were then determined by light scattering, using aNicomp® Model 370 Submicron Particle Sizer.

Example 2

Red Blood Cell ("RBC") Hemolysis Assay

A 4% suspension of red blood cells (RBCs), 0.5 ml, was washed threetimes in PBS and then incubated with free (non-liposomal) etherlipid orliposomal etherlipid, prepared as described above. These samples werevortexed on a 37 deg. C. agitator for 20 hours, and were thencentrifuged for 10 minutes at 3000 rpm. 0.2 ml of the resultingsupernatant was diluted to 1 ml with water, and the percentage hemolysisin the sample was quantitated by spectrophotometric examination at 550nm.

Results from these studies are presented in Table 1 (see below), whereinthe concentration (μM) of edelfosine required to cause 10% RBC hemolysis("HI₁₀ ") in each formulation is set forth. The table's first column isa short-hand designation of the particular formulation, "ELL" standingfor "etherlipid liposome." The second column indicates the components ofthe formulation tested, including dioleoyl phosphatidylethanolamine"(DOPE"), cholesterol ("CHOL"),dioleoyl-phosphatidylethanolamine-glutaric acid ("DOPE-GA"), dioleoylphosphatidylcholine ("DOPC"), palmitoyloleoyl phosphatidylcholine("POPC"), distearoyl phosphatidylcholine "DSPC") egg phosphatidylcholine("EPC") and edelfosine ("EL," for etherlipid). The respective molarratios of the various lipid components are also set forth. The last rowof the table gives the HI₁₀ value for edelfosine alone, i.e., notincorporated in a liposome.

                  TABLE 1                                                         ______________________________________                                        Formulation                                                                              Composition      HI.sub.10                                         ______________________________________                                        ELL 20     DOPE:CHOL:DOPE-GA:EL                                                                           1726 ± 160                                                4312                                                               ELL 12     DOPC:CHOL:DOPE-GA:EL                                                                           670 ± 60                                                  4312                                                               ELL 40     POPC:CHOL:DOPE-GA:EL                                                                           65 ± 6                                                    4312                                                               ELL 28     DSPC:CHOL:DOPE-GA:EL                                                                           32 ± 3                                                    4312                                                               ELL 25     DOPE:CHOL:DOPE-GA:EL                                                                           537 ± 50                                                  3313                                                               ELL 30     EPC:CHOL:DOPE-GA:EL                                                                            314 ± 30                                                  4312                                                               Edelfosine --                5 ± 1                                         ______________________________________                                    

Example 3

Fluorescence Spectroscopy

Liposomes were prepared as described above, and in the presence of anaqueous solution of 0.1 M 6-carboxyfluorescein ("CF"); free CF was thenremoved by gel filtration. CF efflux from liposomes over time wasmonitored by measuring, at 520 nm (excitation at 490 nm), increases inCF fluorescence in the aqueous phase external to the liposomes, upontheir incubation in PBS at 48 deg. C. Fluorescence values, presented inFIG. 1 herein, are expressed as a percentage increase in CF fluorescencerelative to the total CF fluorescence found after disrupting liposomeswith Triton X-100.

FIG. 2 herein compares hemolytic activity and CF leakage in variousliposomal formulations described in Table 1, upon incubation of theliposomes in PBS at 48 deg. C. for 25 minutes. FIG. 3 compares the timerequired for 50% CF leakage in various liposomal formulations, upontheir incubation in 0.5% serum at 37 deg. C.

REFERENCES CITED

U.S. Patent Documents

4,159,988, 4,163,748, 4,235,871, 4,382,035, 4,522,803, 4,588,578,4,734,225, 4,804,789, 4,837,028, 4,920,016, 4,975,282, 5,008,050,5,013,556, 5,030,453, 5,059,421, 5,077,056, 5,169,637, 3,752,886

Foreign Patent Documents

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What is claimed is:
 1. A liposome having a lipid bilayer whichcomprises: (a) an unsaturated or partially unsaturated phospholipid; (b)a sterol; (c) a headgroup-derivatized lipid comprising aphosphatidylethanolamine linked at the ethanolamine group to adicarboxylic acid; and, (d) an etherlipid having the formula: ##STR5##wherein: R1 is Y₁ Y₂ ;Y₂ is CH₃ or CO₂ H; Y₁ is (CH₂)_(n1) (CH═CH)_(n2)(CH₂)_(n3) (CH═CH)_(n4) (CH₂)_(n5) (CH═CH)_(n6) (CH₂)_(n7) (CH═CH)_(n8)(CH₂)_(n9) ; the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer offrom 3 to 23; n1 is zero or an integer of from 1 to 23; n3 is zero or aninteger of from 1 to 20; n5 is zero or an integer of from 1 to 17; n7 iszero or an integer of from 1 to 14; n9 is zero or an integer of from 1to 11; and, each of n2, n4, n6 and 8 is independently zero or 1; Z isoxygen or sulfur; R₂ is an alkyl group or a halogen-substituted alkylgroup having the formula (C(X₁)_(n10) (X₂)_(n11))_(n12) CX₃ X₄ X₅ ; eachof X₁, X₂, X₃, X₄ and X₅ is independently H or a halogen atom; each ofn10 and n11 is independently equal to zero, 1 or 2; n12 is zero or aninteger of from 1 to 23; when n12 is not equal to zero, the sum ofn10+n11 is equal to 2; and, the headgroup-derivatized lipid comprisesfrom about 5 mole percent to about 20 mole percent of the lipid bilayerand the etherlipid comprises from greater than about 10 mole percent toless than about 30 mole percent of the lipid bilayer.
 2. The liposome ofclaim 1 which is a unilamellar liposome having a diameter of fromgreater than about 50 nm to less than about 200 nm.
 3. The liposome ofclaim 1, wherein the phospholipid is a phosphatidylethanolamine or aphosphatidylcholine.
 4. The liposome of claim 3, wherein thephospholipid is a phosphatidylcholine.
 5. The liposome of claim 4,wherein the phosphatidylcholine is dioleoyl phosphatidylcholine.
 6. Theliposome of claim 1, wherein the sterol is cholesterol.
 7. The liposomeof claim 1, wherein the headgroup-derivatized lipid comprises aphosphatidylethanolamine selected from the group consisting ofdipalmitoyl phosphatidylethanolamine, palmitoyloleoylphosphatidylethanolamine and dioleoyl phosphatidylethanolamine.
 8. Theliposome of claim 7, wherein the phosphatidylethanolamine is dioleoylphosphatidylethanolamine.
 9. The liposome of claim 1, wherein theheadgroup-derivatized lipid comprises a dicarboxylic acid selected fromthe group consisting of glutaric acid, sebacic acid, succinic acid andtartaric acid.
 10. The liposome of claim 9, wherein the dicarboxylicacid is glutaric acid.
 11. The liposome of claim 1, wherein theheadgroup-derivatized lipid comprises dioleoyl phosphatidylethanolaminelinked to glutaric acid.
 12. The liposome of claim 1, wherein Y₂ is CH₃.13. The liposome of claim 1, wherein Y₁ is (CH₂)_(n1).
 14. The liposomeof claim 13, wherein Y₁ is (CH₂)₁₇.
 15. The liposome of claim 1, whereinZ is O.
 16. The liposome of claim 1, wherein R₂ is selected from thegroup consisting of CH₃, CH₂ F, CHF₂, CF₃, CH₂ CH₃, CH₂ CF₃ and CF₂ CF₃.17. The liposome of claim 16, wherein R₂ is CH₃.
 18. The liposome ofclaim 1, wherein Y₁ is (CH₂)₁₇, Y₂ is CH₃, Z is O, R₂ is CH₃ and theetherlipid has the formula: ##STR6##
 19. The liposome of claim 18,wherein the phosphatidylcholine is dioleoyl phosphatidylcholine, thesterol is cholesterol, and the headgroup-derivatized lipid comprisesdioleoyl phosphatidylethanolamine linked to glutaric acid.
 20. Theliposome of claim 19, wherein the dioleoyl phosphatidylcholine comprisesabout 40 mole %, cholesterol comprises about 30 mole %, dioleoylphosphatidylcholine comprises about 10 mole % and the etherlipidcomprises about 20 mole % of the bilayer's lipid component.
 21. Theliposome of claim 1, comprising an additional bioactive agent.
 22. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and the liposome of claim 1.